Description: Angiopoietin-1 (ANGPT-1) is a secreted glycoprotein that was first characterized as a ligand of the Tie2 receptor. In a previous study using microarray analysis, we found that the expression of ANGPT-1 was upregulated in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cell lines compared with that in uninfected Burkitt and other leukemia cell lines. Other authors have also reported focal expression of ANGPT-1 mRNA in biopsy specimens of Kaposi's sarcoma (KS) tissue from patients with AIDS. Here, to confirm these findings, we examined the expression and secretion levels of ANGPT-1 in KSHV-infected PEL cell lines and address the mechanisms of ANGPT-1 transcriptional regulation. We also showed that ANGPT-1 was expressed and localized in the cytoplasm and secreted into the supernatant of KSHV-infected PEL cells. Deletion studies of the regulatory region revealed that the region encompassing nucleotides –143 to –125 of the ANGPT-1 -regulating sequence was responsible for this upregulation. Moreover, an electrophoretic mobility shift assay and chromatin immunoprecipitation, followed by quantitative PCR, suggested that some KSHV-infected PEL cell line-specific DNA-binding factors, such as OCT-1, should be involved in the upregulation of ANGPT-1 in a sequence-dependent manner. IMPORTANCE We confirmed that ANGPT-1 was expressed in and secreted from KSHV-infected PEL cells and that the transcriptional activity of ANGPT-1 was upregulated. A 19-bp fragment was identified as the region responsible for ANGPT-1 upregulation through binding with OCT-1 as a core factor in PEL cells. This study suggests that ANGPT-1 is overproduced in KSHV-infected PEL cells, which could affect the pathophysiology of AIDS patients with PEL.

Description: The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product -hydroxy- N -methyl- l -Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo , the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of se Fdx and se FdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N -cetyl- N , N , N -trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.

Description: Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.

Description: Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

Description: Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum β-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type β-lactamase ( bla ) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the bla CTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three bla KPC and one bla IMP ). Overall, pAmpC ( bla ACT/MIR and bla CMY ) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the bla CTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring bla KPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying bla ACT/MIR contained plasmid replicon FIIA. We found that β-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further.

Description: Casein kinase 1, known as DOUBLETIME (DBT) in Drosophila melanogaster , is a critical component of the circadian clock that phosphorylates and promotes degradation of the PERIOD (PER) protein. However, other functions of DBT in circadian regulation are not clear, in part because severe reduction of dbt causes preadult lethality. Here we report the molecular and behavioral phenotype of a viable dbt EY02910 loss-of-function mutant. We found that DBT protein levels are dramatically reduced in adult dbt EY02910 flies, and the majority of mutant flies display arrhythmic behavior, with a few showing weak, long-period (~32 h) rhythms. Peak phosphorylation of PER is delayed, and both hyper- and hypophosphorylated forms of the PER and CLOCK proteins are present throughout the day. In addition, molecular oscillations of the circadian clock are dampened. In the central brain, PER and TIM expression is heterogeneous and decoupled in the canonical clock neurons of the dbt EY02910 mutants. We also report an interaction between dbt and the signaling pathway involving pigment dispersing factor (PDF), a synchronizing peptide in the clock network. These data thus demonstrate that overall reduction of DBT causes long and arrhythmic behavior, and they reveal an unexpected role of DBT in promoting synchrony of the circadian clock network.

Description: Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistant M. pneumoniae has been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistant M. pneumoniae infection in adults. In this study, we survey the macrolide-resistant M. pneumoniae in adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated for M. pneumoniae . All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistant M. pneumoniae isolates were also investigated in vitro . Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) of M. pneumoniae strains isolated from adults with CAP were resistant to erythromycin (MIC = 128 to 〉256 μg/ml), clarithromycin (MIC = 128 to 〉256 μg/ml), and azithromycin (MIC = 32 to 〉64 μg/ml). Furthermore, all macrolide-resistant M. pneumoniae strains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistant M. pneumoniae infection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistant M. pneumoniae in the future.

Description: We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro . Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo . The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN--, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN--, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. IMPORTANCE It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

Description: Our study aims to identify the clinical breakpoints (CBPs) of second-line drugs (SLDs) above which standard therapy fails in order to improve multidrug-resistant tuberculosis (MDR-TB) treatment. MICs of SLDs were determined for M. tuberculosis isolates cultured from 207 MDR-TB patients in a prospective cohort study in China between January 2010 and December 2012. Classification and regression tree (CART) analysis was used to identify the CBPs predictive of treatment outcome. Of the 207 MDR-TB isolates included in the present study, the proportion of isolates above the critical concentration recommended by WHO ranged from 5.3% in pyrazinamide to 62.8% in amikacin. By selecting pyrazinamide as the primary node (CBP, 18.75 mg/liter), 72.1% of sputum culture conversions at month four could be predicted. As for treatment outcome, pyrazinamide (CBP, 37.5 mg/liter) was selected as the primary node to predict 89% of the treatment success, followed by ofloxacin (CBP, 3 mg/liter), improving the predictive capacity of the primary node by 10.6%. Adjusted by identified confounders, the CART-derived pyrazinamide CBP remained the strongest predictor in the model of treatment outcome. Our findings indicate that the critical breakpoints of some second-line drugs and PZA need to be reconsidered in order to better indicate MDR-TB treatment outcome.

Description: The aim of this study was to investigate the epidemiology of pyrazinamide (PZA) resistance and the associated risk factors as well as to evaluate the pncA gene loci as a marker for PZA resistance in China. A population-based multicenter study of pulmonary tuberculosis (TB) cases was carried out from 2011 to 2013 in four Chinese districts/counties with different geographic and socioeconomic features. Testing for multidrug-resistant tuberculosis (MDR-TB) and susceptibility to PZA was done by the proportion method on Lowenstein-Jensen medium and Bactec MGIT 960, respectively. Mutations in the pncA gene were identified by sequencing. Among 878 culture-positive cases, 147 (16.7%) were resistant to PZA, with a significantly higher proportion among MDR isolates than among the first-line drug-susceptible isolates (30.2% versus 7.7%; P 〈 0.001). In total, 136 isolates had a nonsynonymous pncA mutation, with a comparable diagnostic performance between Beijing family and non-Beijing family as well as between MDR-TB and first-line drug-susceptible TB. Furthermore, the mutations in isolates with high-level PZA resistance (MIC 〉 500 mg/liter) were observed mainly in three regions of the pncA gene (codons 51 to 76, codons 130 to 142, and codons 163 to 180). Patients with prior treatment history had a significantly higher risk for PZA monoresistance (odds ratio [OR], 2.86; 95% confidence interval [CI], 1.363 to 6.015) and MDR PZA resistance (OR, 6.47; 95% CI, 3.186 to 13.15), while the additional factors associated with MDR PZA resistance were the patient's age (OR, 1.02; 95% CI, 1.003 to 1.042), lung cavity (OR, 2.64; 95% CI, 1.296 to 5.391). These findings suggest that it is a priority to identify PZA resistance in MDR-TB and that a rapid molecular diagnostic test based on pncA mutations in the Chinese settings where MDR-TB prevalence is high should be developed.